Use of imine-forming polysaccharides as adjuvants and immunostimulants

ABSTRACT

The present invention relates to polysaccharide conjugates that comprise: a polysaccharide that binds to surface-receptors present on Antigen Presenting Cells, conjugated to one or more compounds having stable carbonyl groups covalently attached, either directly or via a bifunctional linker. The conjugates are useful as immuno-stimulants and adjuvants.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of earlier filed U.S.provisional patent application no. 60/060,786, filed Oct. 3, 1997, andis a divisional of U.S. patent application 09/165,310, filed Oct. 2,1998, the contents of which are fully incorporated by reference herein.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention is related to the use of polysaccharidederivatives in vaccines and immunostimulating compositions. Theadjuvants are derivatives of polysaccharides recognized by antigenpresenting cells (APCs).

[0004] 2. Related Art

[0005] Adjuvants have utility in activating the immune system toincrease the efficacy of preventative and therapeutic vaccines.Immunoadjuvants have applications in: (1) the non-specific stimulationof host resistance against infection and cancer, (2) the potentiation ofpreventative vaccine immunogenicity, and (3) the potentiation oftherapeutic vaccine immunogenicity. These adjuvants may preferentiallyenhance cell-mediated immune responses (T cell responses, delayedhypersensitivity), humoral responses (B cell responses, antibodyproduction), or both. Stimulation of humoral immunity is important forprevention of bacterial infections, some viral infections, as well as intherapy of circulating cancers. Cellular immunity is of major importancefor solid tumor cancer therapy and some viral diseases.

[0006] After an initial stimulation by a foreign agent or antigen (suchas viruses, bacteria, or parasites), the immune system usuallyrecognizes and reacts to the agent with an accelerated response uponre-exposure. This enhanced response forms the basis for the enormoussuccess of vaccination for disease prevention. However, the initialimmune response to a foreign antigen requires several days for fullresponse, which is insufficient for protection against infections byhighly virulent organisms. A way to achieve a faster protective immuneresponse is by vaccination or immunization with a pathogen, which isusually attenuated or dead. However, in many cases immunization withkilled microorganisms or with pure antigens elicits a poor short termimmune response with weak or no cell-mediated immunity produced at all.In many cases this poor immune response can be modified by the additionof adjuvants to the antigen preparation. Several polysaccharides(carbohydrate polymers) of mannose (e.g. mannans), β(1,3) glucose (e.g.glucans), β(1,4) acetylated mannose (acemannans), β(1,4)N-acetyl-glucosamine (chitins), and heteropolysaccharides, such asrhamnogalacturonans (pectins), have been shown to stimulate the immunesystem. Antigen presenting cells (APCs) have specificcell-surface-receptors which recognize and bind the sugar moieties ofthese and other polysaccharides. Antigen presenting cells (APCs), suchas dendritic cells and some macrophages, are responsible for taking upantigens and processing them to small peptides in endolysosomes.Processed antigens are expressed on the surface of APCs in conjunctionwith class II MHC. Specifically, reactive T cells recognize antigen andclass II MHC simultaneously, yielding immune responses that are class IIMHC restricted. B cells are stimulated by processed antigens to produceantibodies. These APC surface-receptors (such as the macrophage mannosereceptor and its homologous receptor DEC-205 from dendritic cells) aretransmembrane proteins that mediate endocytosis and apparently play arole in the process of antigen presentation (Stahl, P. D., CurrentOpinion in Immunology 4:49 (1992); Jiang, W., et al., Nature 375:151(1995)). Binding of these polysaccharides to such receptors apparentlyinduces immunostimulation, as shown by the increase in phagocytosis,proliferative responses, release of cytokines, and other activities ofthe immune system. Because of this immunostimulatory activity, thesepolysaccharides have been proposed as vaccine adjuvants.

[0007] Polysaccharide adjuvants exert an immunomodulating effect bymodifying cytokine production, such as upregulating IL-1, and causing amoderate Th1 response. The immune response produced by the Th1 subset ofCD4⁺T cells induces complement fixing antibodies as well as strong,delayed-type hypersensitivity (DTH) reactions associated with γ-IFN,IL-2 and IL-12. Polysaccharides' effects on the native proteinconformation are moderate, preserving the conformational epitopesnecessary to elicit a neutralizing antibody response. However, becausethese adjuvants cannot allow exogenous antigens to be processed via theendogenous pathway, they do not induce a cytotoxic T lymphocyte (CTL)response. Because APCs have cell-surface-receptors specific for certaincarbohydrate moieties, the targeting and delivery to these cells ofantigens associated with these sugar moieties can be significantlyenhanced. Apparently, the role of sugar moieties in the targeting ofantigen delivery is not limited to polysaccharide adjuvants. Forinstance, the modification of quillajasaponin carbohydrate side-chainsby periodic acid oxidation results in a loss of their adjuvanticity.Presumably, this results because of the loss of their targetingcapacity.

[0008] Although the adjuvant properties of certain polysaccharides havebeen known for some time, their use has been largely limited to researchapplications. For instance, it has been shown that glucans can induce ananti-tumor response in mice, and have a preventive effect on acutesepsis. These effects are dependent on the glucans' molecular weight andtheir degree of branching. Mannans are other polysaccharides withadjuvant activity which presumably exert their effect after binding tothe macrophage mannose cell-surface-receptor. Recently, it has beenshown that conjugation of a protein antigen to mannan under oxidizingconditions resulted in a cell-mediated immune response (Apostolopoulos,V. et al., Vaccine 14: 930 (1996)). However, protein antigens conjugatedto mannans under non-oxidative conditions, i.e. without aldehydeformation, elicited only humoral immunity (Okawa, Y. et al., J. Immunol.Meth. 142:127 (1992)) and (Apostolopoulos, V. et al., Proc. Natl. Acad.Sci. USA. 92:10128 (1995)). Stimulation of T-cell immunity has also beenachieved by generating with galactose oxidase under experimentalconditions, aldehydes in the galactosyl residues of cell-surfacepolysaccharides (Zeng, B., et al., Science 256:1560 (1992)). However,this immunostimulation was not reproducible (Rhodes, J. Immunol. Today17:436 (1996)). These results highlight the problems associated withaldehyde instability and/or the inefficient production of aldehydes byenzymatic oxidation.

[0009] It is clinically and economically important for the vaccineindustry to have new and effective adjuvants. The development of noveladjuvants that target antigen presenting cells and provideco-stimulatory signals to stimulate T-cell immunity is the subject ofthe present patent disclosure.

SUMMARY OF THE INVENTION

[0010] The present invention is directed to chemical conjugates (hereinreferred to as polysaccharide conjugates) that comprise (i) apolysaccharide capable of binding to the cell surface of AntigenPresenting Cells (APCs), and (ii) one or more molecules having a stablecarbonyl group (i.e. an aldehyde or ketone group that is capable ofreacting with amino groups to form an imine or Schiff base) whereinmolecules (ii) are attached to the polysaccharide (i) through (iii) adirect covalent bond or covalently via a bifunctional linker in a mannerthat keeps the stable carbonyl group intact. The molecules having animine-forming carbonyl group can be an aromatic or non-aromatic cyclic,aromatic or non-aromatic heterocyclic or non-cyclic compound.Preferably, aromatic or heteroaromatic ketones and aldehydes areemployed as molecules (ii).

[0011] In a second aspect of the invention one or more molecules havinga stable carbonyl group (i.e. an aldehyde or ketone group that iscapable of reacting with amino groups to form an imine or Schiff base)are covalently attached, either directly or via a bifunctional linkingmolecule, to non-adjuvant carbohydrate antigens to provide intrinsicadjuvanticity to the non-adjuvant carbohydrate antigens. The conjugationof one or more molecules having a stable carbonyl group to saidcarbohydrate antigens results in a product having increased efficacy ofpreventive vaccinations compared to the non-conjugated carbohydrateantigens.

[0012] The present invention is directed to enhancing the potentiationof an immune response in a mammal, comprising administering an effectiveamount of a polysaccharide conjugate of the present invention to enhancethe immune response of a mammal to one or more antigens.

[0013] The present invention is also directed to a method ofvaccination, comprising administering one or more antigens, and apolysaccharide conjugate of the present invention.

[0014] The present invention is also directed to pharmaceutical andveterinary compositions comprising one or more of the polysaccharideconjugates of the present invention, and one or more pharmaceuticallyacceptable diluents, carriers or excipients. These compositions may beemployed as immunopotentiators in animals and humans.

[0015] The present invention is also directed to vaccines comprising oneor more antigens, and a polysaccharide conjugate of the presentinvention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present invention is directed to polysaccharide conjugates,comprising:

[0017] (i) a polysaccharide capable of binding the surface of AntigenPresenting Cells (APCs); and

[0018] (ii) one or more molecules having a stable carbonyl group (i.e.an aldehyde ketone group that is capable of reacting with amino groupsto form an imine or Schiff base, also referred to as an “imine-formingcompound”);

[0019] wherein polysaccharide (i) is attached (iii) through a directcovalent bond, or covalently via the residue of a bifunctional linker tosaid one or more molecules (ii). The compounds having the imine-formingcarbonyl group can be an aromatic or non-aromatic cyclic, aromatic ornon-aromatic heterocyclic or non-cyclic compounds. Preferably, aromaticor heteroaromatic ketones and aldehydes are employed as (ii).

[0020] In order to more clearly explain this aspect of the presentinvention, polysaccharide conjugates can be represented by the FormulaI:

P−(L−I)  I

[0021] or pharmaceutically acceptable salts thereof, where

[0022] P is a polysaccharide that is capable of binding to the cellsurface of an Antigen Presenting Cell;

[0023] each L is independently a covalent bond, or the residue of abifunctional linking molecule;

[0024] each I is an imine-forming molecule. Preferred imine-formingmolecules are residues of aromatic or heteroaromatic compounds having(a) a ketone or aldehyde functionality; and (b) a second functionalgroup that is capable of reacting with a complementary functional grouppresent on said polysaccharide or said bifunctional linking molecule, ifpresent; and

[0025] x is greater than or equal to one. The value of x will bedetermined by the number of reactive groups that are covalently modifiedon the polysaccharide.

[0026] A number of factors and strategies will influence the value of x,as will be more fully detailed herein. Generally, x will be a functionof the number of reactive hydroxyl, terminal end hemiacetal, carboxyland/or amine groups that are present on the polysaccharide. Because ofthe diverse molecular weight distribution of useful polysaccharides (P),the degree of modification as expressed by x is expressed as the numberof imine-forming groups introduced per hundred glycosyl residues. Usingthis convention, the value of x can vary from 1 to more than 100, with apreferred range of from 1 to about 50 imine-forming groups per 100glycosyl residues.

[0027] The ratio of imine-forming molecules varies broadly dependingupon the conjugation strategy employed. Control of this ratio is furtherdescribed herein.

[0028] A free hydroxyl, terminal end hemiacetal, carboxylic acid oramine group of the polysaccharide is employed to covalently link thepolysaccharide (P) to either (L) or (I). One or more of these reactivegroups that are present on the polysaccharide can be first “activated”(as further described herein) to increase the reactivity of thesegroups, or the polysaccharide can be reacted with an imine-formingcompound having an “activated” functional group.

[0029] An additional aspect of the invention is directed to conjugatescomprising one or more compounds having a carbonyl group, wherein saidcompounds are covalently attached, either directly or via the residue ofa bifunctional linking molecule, to non-adjuvant carbohydrate antigensto provide intrinsic adjuvanticity to the non-adjuvant carbohydrateantigens. The conjugation of an imine-forming molecules to saidcarbohydrate antigen results in a product having increased efficacy ofpreventive vaccinations compared to the carbohydrate antigen alone.

[0030] Carbohydrate antigens include polysaccharides, includinglipopolysaccharides and peptidoglycans from streptococci, staphylococci,and other bacteria that are used as vaccine antigens.

[0031] Polysaccharides

[0032] Polysaccharides that can be employed to form conjugates of thepresent invention include any polysaccharide, natural or chemicallymodified, that binds to cell surface receptors on APCs. For purposes ofthe present invention, useful polysaccharides comprise a minimum of twosaccharides, preferably seven or more saccharides, and are unbranched orbranched, and can have a molecular weight of from about 1000 to severalmillion Daltons. Preferred polysaccharides have a molecular weight offrom about 1,000 to about 500,000. The polysaccharides may possesschemical modifications as described herein.

[0033] The term “Antigen Presenting Cells” or the abbreviation “APCs”for purpose of the present invention mean dendritic cells andmacrophages that are responsible for taking up antigens, processing themto small peptides, and expressing them on their surface in conjunctionwith class II MHC for presentation to T and B cells.

[0034] During evolution macrophages and dendritic cells have developedcell surface receptors that recognize the carbohydrate moieties fromdifferent microorganisms. These receptors play a critical role inphagocytosis as well as in pinocytosis, two processes that are involvedin antigen presentation. Polysaccharides recognized by thesecell-surface-receptors would be suitable for the construction of theseadjuvants because such polysaccharides provide an effective mechanismfor APC targeting. In some cases, carbohydrate sequences from bacterial,fungal, and animal origins are shared by plant polysaccharides.

[0035] Thus, plant polysaccharides can provide a practical source ofstarting materials in some instances. Although these adjuvants can beprepared with either soluble or insoluble polysaccharides, the solubleforms are preferred.

[0036] The applications of the present disclosure are in no way limitedto plant polysaccharides. They can be extended to othercarbohydrate-containing compounds from different sources that arerecognized by APCs surface receptors. Examples of these otherpolysaccharides are chitins and dextrans which are of animal andbacterial origin respectively. Examples of suitablecarbohydrate-containing compounds are bacterial teichoic acids and theirderivatives, bacterial lipopolysaccharides, lipid A, and theirderivatives.

[0037] Finally, conjugation of compounds carrying imine-forming carbonylgroups to non-adjuvant carbohydrate-containing products is contemplatedto be useful in providing intrinsic adjuvanticity to these products andto increase the efficacy of preventive immunizations. Examples of theseproducts are polysaccharides from streptococci, staphylococci, and otherbacteria that are used as vaccine antigens.

[0038] Among the preferred polysaccharides that are useful in thepresent invention are: β-glucans; mannans; pectins, and 2-acetamidoglucans. Derivatives, including water-soluble derivatives, of thesepolysaccharides are also useful. See Provisional Appl. No. 60/Q83,106,and Divisional Appl. No. 09/165,310, fully incorporated by referenceherein. Preferred polysaccharides are more fully described below.

[0039] β-Glucans: β-Glucans have a backbone chain of (1→3)-linkedβ-D-glucopyranosyl units which has β-D-glucopyranosyl units attached by(1→6) linkages. They are found in several sources, such as yeast, fungi,algae, and cereals. They have a broad range of molecular weights, i.e.between 5,000 to >500,000, which influence their immunomodulatingproperties. In general, β-glucans of high molecular weight that arerelatively insoluble in water have higher biological activity. However,this lack of solubility has precluded the systemic administration ofglucans. Modification of these polysaccharides by introduction ofanionic groups, such as phosphate, sulfate, carboxyl, and others, hasyielded soluble forms that apparently retain their biologicalactivities.

[0040] Soluble glucans can be prepared by one of the followingprocedures: i) isolation from yeast extracts (Hahn & Albersheim, 1978,Plant Physiol. 66:107), ii) sonication of glucan particles (Januz et al.1986, J. Immunol. 137:327, and iii) introduction of anionic groups toinsoluble glucans by sulfonylation, phosphorylation, carboxymethylation,or sulfation ((Bohn & BrMiller, 1995, Carbohydr. Polym. 28:3), (DiLuzio, U.S. Pat. No. 4,739,046, April 1988)). In β-glucans the onlyreducing glucosyl residue (linked at position 3) is located at theterminus of the backbone chain of (1→3)-linked β-D-glucosyl residues.The glucosyl residues attached by (1→6) linkages to the backbone chaindo not have a free reducing group. The smallest fragment that binds tothe monocyte glucan receptor is a (1→3)-linked β-glucanoheptasaccharide.However, this oligosaccharide does not have immunostimulating activity.

[0041] Mannans: Mannans are linear or branched polysaccharides formedexclusively of mannose. Mannans are found in plants, mold, bacteria andother organisms. In certain plants, linear mannans consist of β-(1→4)linked mannosyl residues, whereas in some yeasts, the mannosyl residuesare linked by α-(1→2) and α-(1→6) linkages. In the branched mannans fromSaccharomyces cerevisiae (baker's yeast), the mannan consists of aα-(1→6) linked mannopyranosyl backbone structure substituted on the O-2atoms by side-chains of α-D-mannopyranosyl,α-D-mannopyranosyl-α-(1→2)-α-D-mannopyranosyl and α-D-mannopyranosylα-(1→3)-α-D-mannopyranosyl-α-(1→2)-a-D-mannopyranosyl. In addition, theS. cerevisiae mannan can also be phosphorylated (Barreto-Bergter and P.A. Gorin, Adv. Carbohydr. Chem. Biochem. 41:67 (1983), Vinogradov, E.,et al., Carbohydr. Res. 307:177 (1998)). Although the ability of S.cerevisiae mannans to stimulate cell-mediated immunity is questionable,they enhance the action of lipopolysaccharides in stimulating T-cellresponses (Ohta, M., et al., Immunology 60:503 (1987)). It appears thatmannans can exert their immunostimulatory effects by binding to themacrophage mannose-binding cell-surface receptors. A derivative ofβ-mannans, the acetylated β-(1→4) polymannose, appears to stimulate theimmune system in a manner similar to mannans.

[0042] Pectic polysaccharides: Several pectic polysaccharides areanticomplementary, and they may have different degrees ofimmunopotentiating activity (Yamada, H., et al., Planta Medica 56:182(1990)). Oxidation of these polysaccharides with periodic acid resultsin a loss of anticomplementary activity on the classical pathway, butincreased activity on the alternative pathway (Yamada, H. and Kiyohara,H., Abstracts of Chinese Medicine 3(1):104 (1989)). The polysaccharidesshowing some imrnmunopotentiating activity and thus, being recognized bycell surface-receptors can be grouped broadly into homogalacturonans,rhamnogalacturonans, arabans, galactans, and arabinogalactans. However,not all of these compounds would have biological activity. In manycases, the activity would be dependent on structure, molecular weight,aggregation state, and other parameters. In general, pecticpolysaccharides are a group of sugar polymers associated with 1,4-linkedα-D-galactosyluronic acid residues. These polysaccharides may haveseveral branched oligosaccharides linked to the backbone'sgalactosyluronic acid residues. From previous studies with saponins andother polysaccharides, branched oligosaccharides appear to be relevantfor adjuvanticity.

[0043] 2-Acetamido glucans: chitin, murein and their derivatives: Chitinis a linear N-acetyl-D-glucosamine (NAG) polymer linked by β-(1→4)linkages that has about 16 percent of its NAG residues deacetylated. Itis widely distributed in nature: it has been found in the exoskeleton ofarthropods and in the cell walls of fungi. This polysaccharide haschains that form extensive intermolecular hydrogen bonds, making itinsoluble in water and in different organic solvents. Removal ofchitin's N-acetyl groups by strong alkali treatment yields chitosan, aβ-(1→4) poly-D-glucosamine water-soluble polycation. Chitosan with 70%of its N-acetyl groups removed (deacetylated chitin), shows asignificant immunostimulating activity (Azuma, I., Vaccine 10: 1000(1992)). To avoid the limitations imposed by its insolubility, severalchitin derivatives that are more soluble in water have been developed,such as glycol chitin (Senzyu, K., et al., J Japan, Agri. Chem. Soc.,23:432 (1950)) and carboxymethyl chitin that may also have immunestimulatory properties. Water-soluble alcohol-insoluble chitodextrinscomposed of heptamers or larger NAG oligosaccharides have been preparedby limited acid hydrolysis (Berger, L. R., et al., Biochim. Biophys.Acta 29:522 (1958)). Murein, the major component of bacterial cellwalls, is a polysaccharide made of β-(1→4) linked NAG, with one of theNAG units substituted at C-3 with an O-lactic acid group by an etherlinkage to yield N-acetyl-D-muramic acid (NAM) forming the repeatingsequence NAG-NAM. Because of the lactic acid residues, isolated mureinsare water-soluble. In the bacterial cell wall, murein is attached tocertain peptides to form a cross-linked peptido-glycan. Because of theirstructural similarities, chitin and murein are recognized by the enzymelysozyme, and apparently also by receptors on the macrophage's cellsurface. These structural similarities, which are also present in glycolchitin, may explain the immunostimulatory properties of chitin and someof its derivatives.

[0044] Molecules Having a Stable Carbonyl Group (Imine-FormingMolecules)

[0045] The second element of the conjugates of the present invention isone or more molecules having a stable carbonyl group (i.e. an aldehydeor ketone group) that is capable of reacting with amino groups to forman imine or Schiff base. The molecules having the imine-forming carbonylgroup can be an aromatic or non-aromatic (saturated or partiallyunsaturated) carbocycle, aromatic or non-aromatic (saturated orpartially unsaturated) heterocycle or a non-cyclic, aliphatic compoundthat may have one or more unsaturated bonds.

[0046] There is evidence that certain aromatic compounds with carbonylgroups are very effective in forming imines or Schiff bases uponreaction with amino groups on certain Th-cell surface receptor(s).Because carbonyl groups attached to aromatic compounds are more stable(whereas aliphatic aldehydes are generally unstable), their derivativestypically have a longer shelf life. Furthermore, the hydrophobiccharacter of the cyclic compounds carrying the carbonyl groups willstrengthen the interactions between cell surface receptors and thepolysaccharide conjugates. Consequently, the compounds to be used tomodify the polysaccharides are preferably aryl or heteroaryl aldehydesor ketones. To facilitate the access of these compounds to the aminogroups on T-cells, it is more preferred that they also have somehydrophilic characteristics.

[0047] Compounds that embody some degree of all of the aforementionedproperties are preferred agents for modifying the polysaccharides.Preferred compounds include mono- and di-substituted C₆₋₁₀ arylaldehydesand C₆₋₁₀ aryl(C₁₋₄)alkylaldehydes, compounds comprising an aryl group,such as phenyl or naphthyl and include a formyl or formyl(C₁₋₄)alkylsubstituent. Preferably, these compounds further include one or twoadditional substituents such as halo, hydroxy, C₁₋₄ alkyl, C₁₋₄hydroxyalkyl, C₁₋₄ alkoxy, trifluoromethyl, or benzyloxy. Suitablevalues include benzaldehyde and naphthaldehyde, substituted by one ortwo of hydroxy and halo. Examples include 2,3-, 2,4-2,5-, and3,4-dihydroxybenzaldehyde, 5-chloro-2-hydroxybenzaldehyde, vanillin,ethyl vanillin, naringenin, 3- and 4-hydroxybenzaldehyde, and4-hydroxyphenylacetaldehyde. A second preferred group is hydroxysubstituted C₁₋₄alkyl (C₆₋₁₀)aryl ketones, such as 2-, 3-, and4-hydroxyacetophenone, and hydroxy substituted aryl ketones such as6-hydroxy-1 ,2-naphthoquinone. A third preferred group includesheteroaryl aldehydes and heteroaryl ketones. Useful heteroaryl groupsare thiophene, furan, benzothiophene, benzofuran, pyridine, quinoline,pyridazine, pyrimidine, pyrazole, imidazole, 1,2,3-triazole,1,2,4-triazole, isoxazole, and oxazole, each having a keto, formyl orformyl(C₁₋₄) alkyl substituent, and preferably including an additionalhalo or hydroxy substituent, if these can be accommodated by availablering carbon atoms. Preferably, furanyl, pyridyl, and indolyl aldehydesand ketones are useful heteroaryl cores. Examples of useful heteroarylaldehydes and ketones include pyridoxal, 2-thiophenecarboxaldehyde, and3-thiophenecarboxaldehyde.

[0048] Another relatively stable group of cyclic compounds that containimine-forming carbonyl groups are triterpenoids and steroids having aketo, formyl or formylalkyl substitution. Examples include androsterone,formyldienolone, progesterone, prednisolone, and other derivatives.

[0049] Bifunctional Linkers

[0050] Bifunctional linkers are well known in the art for variousapplications (Hermanson, G. T., Bioconjugate Techniques, Academic Press1996). A number of bifunctional linkers can be employed to form anattachment between a suitable polysaccharide and a suitableimine-forming compound. “Residue of a bifunctional linker” refers to thestructure that links a stable carbonyl compound to the polysaccharideafter the terminal ends of the bifunctional linker have covalentlybonded to said compound and said polysaccharide.

[0051] Non-limiting examples of linker groups that can be used to linkthe stable carbonyl containing compound to the polysaccharide arealkylene diamines (NH₂—(CH₂)_(n)—NH₂), where n is from 2 to 12;aminoalcohols (HO—(CH₂)_(r)—NH₂), where r is from 2 to 12; aminothiols(HS—(CH₂)_(r)—NH₂), where r is from 2 to 12; and amino acids that areoptionally carboxy-protected; ethylene and polyethylene glycols(H—(O—CH₂—CH₂)_(n)—OH, where n is 1-4). Suitable bifunctional diaminecompounds include ethylenediamine, 1,3-propanediamine,1,4-butanediamine, spermidine, 2,4-diaminobutyric acid, lysine,3,3′-diaminodipropylamine, diaminopropionic acid,N-(2-aminoethyl)-1,3-propanediamine, 2-(4-aminophenyl)ethylamine, andsimilar compounds.

[0052] When a carboxyl group of the polysaccharide is employed as theconjugating group, one or more amino acids can be employed as thebifunctional linker molecule. Thus, an amino acid such as β-alanine orγ-aminobutyric acid, or an oligopeptide, such as di- or tri-alanine canbe employed as a suitable linking molecule.

[0053] Preferred bifunctional linking groups include:

[0054] —NH—(CH₂)_(r)—NH—, where r is from 2-5,

[0055] —O—(CH₂)_(r)—NH—, where r is from 2-5,

[0056] —NH—CH₂—C(O)—,

[0057] —O—CH₂—CH₂—O—CH₂—CH₂—O—,

[0058] —NH—NH—C(O)CH₂—,

[0059]

[0060] —NH—C(CH₃)₂—C(O)—,

[0061] —S—(CH₂)_(r)—C(O)—, where r is from 1-5,

[0062] —S—(CH₂)_(r)—NH—, where r is from 2-5,

[0063] —S—(CH₂)_(r)—O—, where r is from 1-5,

[0064] —S—(CH₂)—CH(NH₂)—C(O)—,

[0065] —S—(CH₂)—CH(COOH)—NH—,

[0066] —O—CH₂—CH(OH)—CH₂—S—CH(CO₂H)—NH—,

[0067] —O—CH₂—CH(OH)—CH₂—S—CH(NH₂)C(O)—,

[0068] —O—CH₂—CH(OH)—CH₂—S—CH₂—CH₂—N—,

[0069] —S—CH₂—C(O)—NH—CH₂—CH₂—NH—, and

[0070] —NH—O—C(O)—CH₂—CH₂—O—P(O₂H)—.

[0071] The preferred combinations of polysaccharide, imine-formingcompound, linkers and ratios for each may include but are not limitedto: Polysaccharide imine-forming linker (P) compounds (I) (L) L/P* I/P*Glucans 4-hydroxy- -NH-(CH₂)_(n)-NH-C(O)- 5-30 5-20 benzaldehyde Glucans4,6-dioxo- -C(O)-NH-(CH₂)_(n)-NH- 5-30 5-20 heptanoic acid Glucanspyridoxal -NH-O-C(O)-(CH₂)-C-O- 5-30 5-20 5-phosphate Glucans2,4-dihydroxy- -NH-(CH₂)_(n)-NH-C(O)- 5-30 5-20 benzaldehyde Glucanspyridoxal -NH-(CH₂)_(n)-NH-C(O)- 5-30 5-20 5-phosphate Glucans2-thiophene- -C(O)-NH-(CH₂)_(n)-NH- 5-30 5-20 carboxaldehyde Glucans3-thiophene- -NH-O-C(O)-(CH₂)-C-O- 5-30 5-20 carboxaldehyde Mannans4-hydroxy- -NH-(CH₂)_(n)-NH-C(O)- 5-30 5-20 benzaldehyde Mannans4,6-dioxo- -C(O)-NH-(CH₂)_(n)-NH- 5-30 5-20 heptanoic acid Mannanspyridoxal -NH-O-C(O)-(CH₂)-C-O- 5-30 5-20 5-phosphate Mannans2,4-dihydroxy- -NH-(CH₂)_(n)-NH-C(O)- 5-30 5-20 benzaldehyde Mannanspyridoxal -NH-(CH₂)_(n)-NH-C(O)- 5-30 5-20 5-phosphate Mannans2-thiophene- -C(O)-NH-(CH₂)_(n)-NH- 5-30 5-20 carboxaldehyde Mannans3-thiophene- -NH-O-C(O)-(CH₂)-C-O- 5-30 5-20 carboxaldehyde Pectic4-hydroxy- -NH-(CH₂)_(n)-NH-(O)- Poly- benzaldehyde saccharides Pecticpyridoxal -NH-(CH₂)_(n)-NH-C(O)- 5-30 5-20 Poly- 5-phosphate saccharidesPectic 2-thiophene- -C(O)-NH-(CH₂)_(n)-NH- 5-30 5-20 Poly-carboxaldehyde saccharides Pectic 3-thiophene- -NH-O-C(O)-(CH₂)-C-O-5-30 5-20 Poly- carboxaldehyde saccharides Murein 4-hydroxy--CH₂-CHOH-(CH₂)-O- 5-30 5-20 benzaldehyde (CH₂)_(n)-O-CH₂- CHOH-CH₂-Murein 4,6-dioxo- -C(O)-(CH₂)_(n)-NH- 5-30 5-20 heptanoic acid Murein2,4-dihydroxy- -CH₂-CHOH-CH₂- 5-30 5-20 benzaldehyde Murein pyridoxal-NH-(CH₂)_(n)-NH-C(O)- 5-30 5-20 5-phosphate Murein 2-thiophene--C(O)-NH-(CH₂)_(n)-NH- 5-30 5-20 carboxaldehyde Murein 3-thiophene--NH-O-C(O)-(CH₂)-C- 5-30 5-20 carboxaldehyde (O)- Glycol chitinpyridoxal -O-C(O)-(CH₂)_(n)-C-O- 5-30 5-20 5-phosphate Glycol chitinpyridoxal -NH-(CH₂)_(n)-NH-C(O)- 5-30 5-20 5-phosphate Glycol chitin2-thiophene- -C(O)-NH-(CH₂)_(n)-NH- 5-30 5-20 carboxaldehyde Glycolchitin 3-thiophene- -NH-O-C(O)-(CH₂)-C- 5-30 5-20 carboxaldehyde (O)-

[0072] Preparation of Imine-Forming Polysaccharide Adjuvants

[0073] The present invention is also directed to processes for thepreparation of polysaccharide conjugates of the present invention.Structure/function studies of adjuvant polysaccharides and saponins,have shown that the integrity of the carbohydrate chains'structures arecritical for their adjuvanticity. Apparently, the recognition of thecarbohydrate moieties by APCs surface-receptors is essential fortargeting of the cells as well as to exert their immunostimulatoryeffects. The adjuvant activity of triterpene saponins also requires analdehyde group in the triterpenoid moiety. It has also been recentlyshown that small organic molecules capable of forming imines orSchiff-bases can provide a co-stimulatory signal to T-cells, thusobviating the need for their stimulation by the B7-1 receptor present onAPCs (Rhodes, J., et al., Nature 377:71 (1995)). Addition of (i) acyclic or heterocyclic aromatic compound, or cyclic compounds havingimine-forming carbonyl groups, to (ii) certain polysaccharidesrecognized and bound by APCs will result in products with superioradjuvant properties. These adjuvant molecules will possessimmunomodulating and targeting properties.

[0074] A. Addition of Imine-Forming Compounds to Terminal ReducingGlycosyl Residues

[0075] A method for covalently attaching imine-forming compounds toterminal reducing glycosyl residues of β-glucans and β-mannans isdescribed herein with reference to Scheme 1.

[0076] Because β-glucans and β-mannans are comprised of either glucosylor mannosyl residues, the functional groups available for chemicalmodifications are largely hydroxyl groups (-OH) with limited reactivity.In addition, there is one terminal reducing glucosyl residue per polymerchain. In general the primary hydroxyl groups of glycosyl residues aremore reactive than the secondary hydroxyl groups. However, it ispossible that the structure of a particular polysaccharide may imposecertain constraints (steric or electronic) on the hydroxy groups'reactivity. This will create a hierarchy of hydroxy groups that couldfavor the production of certain dominant products under limitingreaction conditions.

[0077] The restricted number of terminal reducing sugars in glucans andmannans provides a highly specific site for introduction of new chemicalgroups. This specificity makes the terminal-end glycosyl hemiacetals apreferred group for commercial production of modified polysaccharideadjuvants of the present invention.

[0078] The chemical modifications described here can be used withsoluble or insoluble glucans from different organisms. However, they areprovided only as examples, not as limitations of the syntheticprocedures available. Because the carbohydrate moieties' role in thesenew adjuvants is the targeting of APCs, the useful molecular weightrange can be very broad, i.e. from about a thousand to several millions.In the present invention, soluble oligo- and polysaccharides ofmolecular weights ranging from about 1,000 to several hundred thousandsare preferred.

[0079] The reducing terminus of oligosaccharides provides a selectiveand convenient site for the direct covalent attachment of molecules withamino groups, such as bifunctional diamine compounds. The reductiveamination procedure involves reacting the terminal reducing glycosylresidue(s) in the oligosaccharide (or polysaccharide) with a compoundcarrying one or more primary amino groups in the presence of sodiumcyanoborohydride (NaCNBH₃).

[0080] The cyanoborohydride anion selectively reduces the imine orSchiff base formed by an aldehyde or ketone and an amine. Since only asmall percentage of the time the terminal glucosyl hemiacetals are intheir formyl or open form, the reaction may proceed at very low rate.Because the presence of both imine-forming carbonyls and primary aminesin a molecule would largely result in the production of undesirableproducts, the additions are carried on in a two-step procedure,summarized as follows:

[0081] Step 1. Glucan/mannan oligosaccharides or polysaccharides (1) aresuspended/dissolved in an appropriate solvent, such as aqueousacetonitrile, dimethylformamide (DMF), pyridine, or aqueous bufferscontaining a tertiary amine buffer pH 9.0, and a suitable di aminecompound (2), where n is from about 2 to about 12, preferably, 2 to 4 isadded in the same solvent with the pH adjusted to 9.0. [Suitablebifunctional diamine compounds are ethylenediamine, 1,4-butanediamine,spermidine, 2,4-diaminobutyric acid, lysine, 3,3′-diaminodipropylamine,diaminopropionic acid, N-(2-aminoethyl)-1,3-propanediamine,2-(4-aminophenyl)ethylamine, and similar compounds]. The diaminecompound that is added should be about 5 to 10-fold excess compared tothe molar equivalent of free aldehyde groups in the carbohydrate (i.e.one free aldehyde per carbohydrate polymerchain). Sodiumcyanoborohydride dissolved in 50% acetonitrile is added to the reactionmixture, and the reaction is allowed to proceed at 25° C. with gentlestirring for several days. The amount of amine compound incorporated inthe polysaccharide depends on the reaction conditions as well as theglucan preparation. Therefore, the amount of diamine compoundincorporated is determined daily to establish the reaction time neededto reach the required level of diamine incorporation. The modifiedaminated glucan/mannan (3), containing 1 mole of diamine spacer perpolysaccharide chain) is recovered by precipitation with 6 volumes ofethanol, or other suitable solvent, for 8 hours at 4° C. The precipitateis redissolved in water, is filtered, and is re-precipitated with 5volumes of ethanol for 24 hours at 4° C. The material is dissolved inwater and is lyophilized.

[0082] Step 2. Aromatic cyclic or heterocyclic compounds with animine-forming carbonyl group (4), and one or more hydroxyl groups, suchas 4-hydroxybenzaldehyde, 2,4-dihydroxybenzaldehyde, vanillin, ethylvanillin, naringenin, and other similar compounds are preferred foraddition to the aminated polysaccharides. However, other compoundshaving carbonyl groups such as steroid and triterpenoid derivatives canalso be used. Small aliquots 10 mmol of either 4-hydroxybenzaldehyde(1.2 g), vanillin (1.5 g), or 5-chloro-2-hydroxybenzaldehyde (1.6 g)dissolved in 10 ml of dioxane or acetone are added to 10 mmol (1.6 g) of1,1′-carbonyldiimidazole (carbodiimidazole or CDI) orN,N′-carbonyldiimidazole dissolved in 10 ml of anhydrous dioxane oracetone. The mixture is reacted for 6-8 hours at room temperature withmixing while protected from atmospheric moisture. The reaction productsare a highly reactive intermediate imidazole carbamate (5) which isformed with the hydroxy from the aromatic aldehyde derivatives, plusimidazole.

[0083] Step 3. This reaction mixture can be added to the aminatedpolysaccharides without prior isolation of the intermediate imidazolecarbamate. The carbamate couples with the modified polysaccharides'amino groups to yield stable carbamate linkages. (Imidazole carbamatederivatives can be isolated by chromatography, differential extractions,or other procedures). To minimize reactions of the polysaccharides'hydroxy groups with the imidazole carbamate intermediate, the couplingreaction should take place in the presence of equimolar amounts of aminoand imidazole carbamate groups. The amount of amino groups in theaminated polysaccharide is determined with trinitrobenzenesulfonic acid(TNBS), or is estimated from its content of C, N, H, and O as determinedby elementary analysis. The aminated glucan or mannan is suspended in asuitable anhydrous organic solvent, such as DMF, dioxane, or pyridine,and the pH is adjusted to about 9.5-10 with triethylamine. An aliquot ofthe carbamate intermediate containing an amount equivalent to the aminogroups of the polysaccharide preparation is added, and the reaction isallowed to proceed for 12 to 18 hours at room temperature protected frommoisture. About 6-8 volumes of cold ethanol are added to the reaction toprecipitate the polysaccharide-aromatic aldehyde conjugate (6), which iscollected by filtration. The conjugated polysaccharide is redissolved inwater, and re-precipitated again with 6-8 volumes of ethanol or othersuitable solvent. Efficacy of coupling is determined by one of thefollowing methods: i) measuring the residual amino groups in thepreparation with TNBS, ii) determining spectrophotometrically the amountof aromatic compound present in the preparation, or iii) by directestimation of the aldehyde groups either with Schiff reagent orspectrophotometrically with N-methyl benzothiazolone hydrazone (MBTH)(Paz, M. A., et al., Archiv. Biochem. Biophys. 109:548 (1965)). Thepolysaccharide-aromatic aldehyde conjugate is dissolved in water and islyophilized.

[0084] It is also possible to create new aldehyde groups in thepolysaccharide chain by mild oxidation with periodic acid. Afteroxidation, the polysaccharide with the additional aldehyde groups isprecipitated with alcohol and subjected to reductive amination asdescribed above.

[0085] B. Addition of Imine-Forming Compounds Via the Polysaccharide'sHydroxy Groups

[0086] Another method to prepare glucan or mannan conjugated to carbonylcarrying compounds is to use the polysaccharides hydroxy groups forconjugate formation. This method is illustrated by reference to Scheme2. Because of the number of hydroxy groups per glycosyl residue, thismethod allows the preparation of conjugates with higher densities ofcarbonyl groups. One can activate the polysaccharide's hydroxy groupsand let react with the carbonyl-carrying molecule.

[0087] Conjugation of compounds carrying carbonyl groups to the hydroxylgroups of polysaccharides can be made with N,N′-disuccinimidyl carbonate(DSC). Hydroxyl groups that are activated with DSC react almostexclusively with primary amines, avoiding the cross-linking of thepolysaccharide chains via their —OH groups. Hydroxyl groups of β-1,3glucans are activated with DSC, and subsequently reacted withethylenediamine to introduce amino groups into the glucan. These aminogroups can then react with one of the following N-hydroxysuccinimide(NHS) esters: 3- or 5-formylsalicylic acid-, 4-formylcinnamic acid, and3- or 4-carboxybenzaldehyde, to form an aromatic aldehyde-glucanconjugate. Scleroglucan (7), a β-1,3 glucan with single β-1,6 linkedD-glucose branches on every third glucose unit and with a molecularweight of about 130,000, is dissolved in water (2-5% solution), andlyophilized to yield a powdery product easily suspended in organicsolvents. Two g of the lyophilized scleroglucan (12 mmoles glucosylresidues) are suspended in 40 ml of DMF containing 0.8 g of DSC (3mmoles). To this suspension add in an hour 20 ml of dry pyridinecontaining 0.75 ml of anhydrous triethylamine (5.5 mmoles) and let reactfor an additional 4 hours at room temperature. One volume of acetone isadded to this reaction, and the insoluble activated scleroglucansuccinimidyl carbonate (8) is collected and rinsed by filtration. Theactivated scleroglucan, diss6lved or suspended in 20 ml of water, isslowly added with stirring to 100 ml of 0.2 M K potassium bicarbonatecontaining 2 ml of ethylenediamine (30 mmoles or 10× excess over themaximum polysaccharides' activated —OH groups) and adjusted to pH 8.5.After 4 hours at room temperature, the reaction is concentrated anddialyzed against water to remove excess of reactants from the aminatedscleroglucan derivative (9) and lyophilized. The aminated polysaccharideis then reacted with succinimidyl-3-formylsalicylic acid ester.

[0088] Succinimidyl-3-formylsalicylic acid ester is prepared as follows:to 0.49 g of 3-formylsalicylic acid (3 mmoles) and 0.42 g of NHS (3.5mmoles) dissolved in 10-15 ml of DMF, 1.28 g of CMC or1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide p-toluenesulfonate (3mmoles) are added and left to react for 5 hours at room temperatureprotected from moisture. To this reaction mixture add 2.1 ml of2-mercaptoethanol (30 mmoles) to quench the unreacted CMC, mix and reactfor 10 minutes at room temperature and use thesuccinimidyl-3-formylsalicylic acid ester (10) immediately. (The3-formylsalicylic acid can be replaced by an equimolar amount of5-formylsalicylic acid, 4-formylphenoxyacetic acid or3-carboxybenzaldehyde.)

[0089] To the aminated scleroglucan (about 2 g) dissolved in 25 ml of0.1 M of MOBS (4-[N-morpholino]butanesulfonic acid) buffer, pH 7.6, addwith stirring the DMF containing the succinimidyl 3-formylsalicylic acidester and the mercaptoethanol, and let react with stirring at roomtemperature for 4-6 hours. The 3-formylsalicylic acid-scleroglucanderivative (11) is precipitated with 6-8 volumes of ethanol, andcollected and washed with ethanol to remove excess of reactants. Theprecipitated scleroglucan derivative is dissolved in 50 ml of water,dialyzed against water, and lyophilized. The 3-formylsalicylic acidcontent of the scleroglucan derivative can be determined from its H, C,N, and O elementary composition. The content of aromatic aldehyderesidues in the scleroglucan derivative can also be determinedspectrophotometrically as follows: dissolve 5-8 mg of the scleroglucanderivative in 4 ml of 0.05 M KOH and read the UV spectra between 220 and320 nm using as a blank a solution of equal concentration of unmodifiedscleroglucan in 0.05 M KOH. Calculate the 3-formylsalicylic acidincorporated using the extinction coefficient for this compounddetermined in 0.05 M KOH. The aldehyde content can also be determinedeither calorimetrically with Schiff reagent or spectrophotometricallywith MBTH.

[0090] Conjugation of compounds carrying both carbonyl and hydroxylgroups to polysaccharides, such as mannans, can be carried out byactivating the polysaccharide hydroxyl groups with p-toluenesulfonyl(tosyl) chloride (Nil sson, K., et al., Acta Chem. Scan. 35:19 (1981);Nilsson, K., et al., Methods Enzymol. 104:56-69 (1984)). See Scheme 3-a.

[0091] Because several of the primary hydroxyl groups in mannan from S.cerevisiae are either phosphorylated or linked to phosphoesters, and areunavailable for tosylation, it is necessary to remove the phosphate byalkaline hydrolysis under reducing conditions. Baker's yeast mannan, 4-5g, is dissolved in 200 ml of 1.5 M KOH containing 1% sodium borohydrideand refluxed at 100° C. for 2 hours with constant stirring. After 2hours at 100° C. cool the solution to about 50° C. and neutralize itwith about 18 ml of acetic acid. The neutralized mannan solution isadded with stirring to 1.5 liters of chilled ethanol and the mixture isleft standing for 4 hours to precipitate the mannan. The precipitatedmannan is collected by filtration, dissolved in water, dialyzed againstwater to remove salts and lyophilized.

[0092] One g of lyophilized mannan (12) (6 mmoles mannose), dried bysuspension in pyridine and the azeotrope removed, is resuspended in 15ml of DMF, and mixed with 2.5 g of tosyl chloride (2.15 mmoles)dissolved in 5 ml of DMF. To this mixture add 5 ml of pyridine and reactwith stirring for 18 hours at room temperature, to yield a tosylatedmannan (13) having 1 tosyl group for every 20-25 mannosyl residues. Thedegree of tosylation is determined in a sample of the activated mannanthat has been precipitated and washed with alcohol as follows. Dissolve8 mg of the precipitated mannan in 4 ml of 0.1 N KOH, and measure its UVabsorption spectra (220 to 360 nm) against a blank of unmodified mannan(8 mg in 4 ml of 0.1 N KOH). Determine the content of tosyl groups byusing an extinction coefficient for tosyl of 480 M⁻¹cm⁻¹ at 261 nm. Tothe tosylated mannan (13) preparation (˜0.9 g) resuspended in 20 ml of0.5 M K bicarbonate, add 2 g of 2-cysteamine HCl (18 mmoles) and adjustthe pH with 1 N KOH to 9.5-10. Let react for 24 hours at 40° C. withstirring and under a nitrogen atmosphere to introduce about 1 cysteamineresidue per tosylated —OH. Dialyze the reactions against water to removeexcess of reactants and salts, and lyophilize the mannan-cysteaminederivative (14). The amount of cysteamine bound to the tosylated mannancan be determined from the derivative's H, C, S, O and N elementarycomposition. The cysteamine incorporation can also be estimated bymixing 10 ml of 0.05 M K carbonate buffer pH 9.5 containing 2 mg of theaminated mannan with 1 ml of an aqueous TNBS (7.2 mg/ml) and reactingfor 2 hours at 40° C. Measure the absorbancy at 363 nm against a blankof buffer plus TNBS, and determine the incorporated —NH₂ using a molarextinction coefficient of 11,000 M⁻¹cm⁻¹.

[0093] Scheme 3-b illustrates attachment of an imine-forming compound tomannan-cysteamine. Suspend the lyophilized mannan-cysteamine (0.8-0.9 g)in 20 ml of pyridine, remove the azeotrope under reduced pressure toeliminate water, and resuspend the mannan-cysteamine residue (14) in 40ml of pyridine:tetrahydrofuran (10:1). To this suspension add 0.37 g ofDCC (dicyclohexylcarbodiimide) (1.8 mmole), 0.3 g ofp-carboxybenzaldehyde and 0.21 g of NHS (1.8 mmole) to form in situ thep-carboxybenzaldehyde-NHS ester intermediate (10). Let the reactionproceed for 24 hours with stirring at room temperature. Stop thereaction by adding 100 ml of water to the mixture to accelerate theformation of DCU. After 30 minutes, add an additional 400 ml of water todissolve the p-carboxybenzaldehyde-mannan derivative (15). After 6 hoursof stirring at room temperature, let the dicyclohexyl urea (DCU) settleovernight and remove it by decantation. Filter the supernatant,concentrate it under reduced pressure, dialyze against water andlyophilize the p-carboxybenzaldehyde-mannan derivative (15). Thearomatic aldehyde incorporated to the mannan can be determinedspectrophotometrically as follows. Dissolve 5-8 mg of the mannanderivative in 4 ml of 0.05 M KOH and read the UV spectra between 220 and320 nm using as a blank a solution of equal concentration of unmodifiedmannan in 0.05 M KOH. To calculate the p-carboxybenzaldehydeconcentration use its extinction coefficient determined in 0.05 M KOH.The aldehyde content can also be determined either calorimetrically withSchiff reagent or spectrophotometrically with MBTH.

[0094] C. Addition of Imine-Forming Compounds to Carboxy Groups ofPectic Polysaccharides

[0095] Carboxylic groups from pectic polysaccharides,(homogalacturonans, rhamnogalacturonans, arabinogalactans, arabans, orgalactans), such as galacturonic, glucuronic, aceric, Kdo or3-deoxy-D-manno-octulosonic acid, and other acids, are reactive groupsthat can be used to couple the pectins to carbonyl carrying(imine-forming) compounds. Carboxylic groups can be coupled specificallyto amines by using dicyclohexylcarbodiimide (DCC) andN-hydroxysuccinimide (NHS). See Scheme 4.

[0096] This reaction can be carried out in organic solvents such asdioxane, DMF, pyridine, acetonitrile, or mixtures of the same. Thecoupling reaction can also be carried out in aqueous or semi-aqueousmedia using one of the water-soluble carbodiimides such as1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or CMC, inconjunction with either N-hydroxysulfosuccinimide (sulfo-NHS) or NHS.The number of amine groups per glycosyl residue can be selected by useof a limiting amount of CMC in the presence of an excess of diamineligand. This approach is illustrated in Scheme 4. In one example, to2.0-2.2 g of low methoxyl Na⁺ pectinate (<11 mmoles galacturonic acid)dissolved in 50 ml of hot water, 15 ml of settled strongcation-exchanger polystyrene sulfonic acid resin in H⁺ form (Dowex,50-X8) are added and stirred for 2 hours. The suspension is filtered toremove the resin, and the resin is washed with 20 ml of warm water. Thepectin (H⁺ form) (16) solution is reduced in volume by partiallyophilization, low-pressure rotary evaporation, or reverse osmosis, toapproximately 40 ml. To the pectin solution (H⁺ form) add pyridine tobring the pH to ˜7.6. To this solution 1 g of CMC (2.35 mmoles) and 0.41g of NHS (3.4 mmoles) dissolved in 10 ml of pyridine and 2 ml ofethylenediamine (30 mmoles) are added with stirring, and the mixture isleft to react overnight at room temperature with constant stirring.Separation of the aminated polysaccharide (17) from the other reactantsis accomplished by precipitating it with 6-8 volumes of ethanol. Theethanol-washed, precipitated, aminated pectin is dissolved in water,dialyzed and lyophilized to yield a powdery product easily dissolved inwater, and its amino group content determined with TNBS. The aminatedpectin is then reacted with succinimidyl-3-formylsalicylic acid esterprepared as follows. To 0.49 g of 3-formylsalicylic acid (3 mmoles) and0.42 g of NHS (3.5 mmoles) dissolved in 10-15 ml of DMF, 1.28 g of CMC(3 mmoles) are added and left to react for 5 hours at room temperatureprotected from moisture. To this reaction mixture 2.1 ml of2-mercaptoethanol (30 mmoles) are added to quench the unreacted CMC, andafter 10 minutes at room temperature the formedsuccinimidyl-3-formylsalicylic acid ester (10) is used immediately. (The3-formylsalicylic acid can be replaced by an equimolar amount of3-carboxybenzaldehyde, 4-formyl-phenoxyacetic acid or 4,5-dioxoheptanoicacid).

[0097] To 2 g of the aminated pectin, dissolved in 30 ml of 0.1 M MOBS(4-[N-morpholino]butanesulfonic acid) buffer, pH 7.6, add the reactionmixture in DMF containing the succinimidyl 3-formylsalicylic acid esterand the mercaptoethanol, and let react with stirring at room temperaturefor 6 hours. The 3-formylsalicylic acid-pectin derivative (18) isprecipitated with 6-8 volumes of ethanol, and then collected and washedwith ethanol to remove excess of reactants. The precipitated pectinderivative is dissolved in a minimal volume of water, dialyzed againstwater, and lyophilized. The aminated pectin can also be reacted with asuccinimidyl carbonate intermediate of a hydroxylated,carbonyl-containing compound, such as 4-hydroxyphenylacetaldehyde,6-hydroxy-2-naphthoquinone, 4-hydroxybenzaldehyde,2,4-dihydroxybenzaldehyde, formyldienolone, progesterone, androsterone,prednisolone, or pyridoxal. After mixing the reaction with 6 volumes ofethanol, the aldebyhe-containing pectic polysaccharide derivative isthen recovered by filtration, dissolved in water, dialyzed against waterand lyophilized.

[0098] Alternatively, the aminated pectin (17) can be reacted with acompound containing both a free and a protected aldehyde group, liketerephthaldehyde monodiethylacetal. The aminated pectin is first reactedwith the free aldehyde group to form a stable bond, and subsequently theprotected aldehyde is released by mild acid hydrolysis, as describedhere. To 2 g of aminated pectin suspended in 30 ml of aqueoustetrahydrofuran or another suitable solvent, add 0.43 g ofterephthaldehyde monodiethylacetal (2 mmoles), 0.18 g of sodiumcyanoborohydride (3 mmoles) and let react overnight with stirring at 40°C. to yield the terephthaldehyde monodiethylacetal derivative of pectin(19). The pectin derivative is precipitated with 6 volumes of ethanol,collected, and washed by filtration with more ethanol. Theterephthaldehyde monodiethylacetal pectin derivative is dissolved in aminimum volume of 0.05 M HCl and heated at 100° C. for 15-20 minutes toconvert the acetal to the aldehyde form. After hydrolysis the solutionof phenylacetaldehyde pectin derivative (20) is brought to neutralitywith NaOH dialyzed against water and lyophilized. The aromatic aldehydecontent of the pectin derivatives is determined spectrophotometricallyas follows: dissolve 5-8 mg of the conjugated pectin in 3 ml of 0.05 NNaOH and scan the UV spectra between 220 and 320 nm against a blanksolution containing the same amount of unmodified pectin. The aromaticaldehyde concentration of the pectin derivative is calculated using theextinction coefficient of the free aromatic aldehyde as determined in0.05 M NaOH. The aldehyde content can also be determinedspectrophotometrically with MBTH or calorimetrically with Schiffreagent.

[0099] Alternatively, the introduction of carbonyl containing compoundsto pectic polysaccharides can be carried out in organic solvents. In oneexample, CMC, NHS, and a 10-fold excess of a diamine with respect to CMCare added to anhydrous lyophilized pectin (H⁺ form) suspended inDMF-pyridine (6:4, v/v) and the mixture is left to react with stirringat room temperature overnight. The number of amine groups per glycosylresidue can be selected by using a limiting amount of CMC in thepresence of an excess of diamine ligand. Separation of the aminatedpolysaccharide from the other reactants is accomplished by precipitatingit with 6-8 volumes of ethanol. The ethanol-washed, precipitated,aminated pectin is dissolved in water and lyophilized to yield a powderyproduct easy to resuspend in DMF-pyridine. The aminated pectin isreacted in DMF-pyridine with either (i) a succinimidyl ester of acarboxylated carbonyl-containing compound, such as 4,5-dioxoheptanoicacid, 3- or 5-formylsalicylic acid, 4-formylcinnamic acid, or 3- or4-carboxybenzaldehyde, or (ii) the succinimidyl carbonate intermediatesof a hydroxylated, carbonyl-containing compound, such as2,4-dihydroxybenzaldehyde, 4-hydroxybenzaldehyde,4-hydroxyphenylacetaldehyde, 4′-hydroxyacetophenone,6-hydroxy-2-naphthoquinone, formyldienolone, progesterone, androsterone,prednisolone, pyridoxal. The aldehyde-containing pectic polysaccharidederivative is then recovered by filtration after mixing the reactionwith 6 volumes of ethanol, dissolved in water, dialyzed against waterand lyophilized.

[0100] D. Addition of Imine-Forming Compounds to Amino Groups of ChitinDerivatives

[0101] The insolubility of chitin in most solvents hinders the additionof imine-forming compounds to it. However, introduction of carbonylcarrying compounds into the water-soluble chitosan yieldswater-insoluble gels, which are presumably the result of cross-linkingby Schiff-bases between the glucosamine's amine groups and the carbonylgroups from different polysaccharide chains. These obstacles are avoidedby use of water-soluble chitin derivatives in which about 85% of theamine groups are N-acetylated, such as glycol chitin (6-O-hydroxypropylchitin or 6-O-hydroxyethyl chitin) in which primary hydroxyls of theglucosamine are hydroxyethylated. Scheme 5 illustrates this procedure.

[0102] In one embodiment, glycol chitin is prepared as follows. To 5 gof finely ground chitin (22) (equal to 21.25 mmoles of N-acetylglucosamine and 3.75 mmoles glucosamine) suspended in 30 ml of 42% NaOHfor 2 hours at room temperature, add with stirring 70 ml of an ice-watermixture to yield a highly viscous dispersion of alkaline-chitin. To thisdispersion, 5 g of ethylene oxide (114 mmoles) are added by bubblinginto the solution while stirring for 1-2 hours at 30-35° C. The glycolchitin (23) is then precipitated with 6 volumes of 80% ethanol, washedon glass filter paper with 80% ethanol, dissolved in water, dialyzedagainst water, lyophilized and the content of free amine groupsdetermined calorimetrically with TNBS. To 1.7-2 g of lyophilized gycolchitin (about 1.5 mmoles of glucosamine) dissolved/suspended in 40 ml ofwater add 40 ml of pyridine containing 1.60 g of terephthaldehydemonodiethylacetal (7.5 mmoles), 0.4 g sodium cyanoborohydride (6 mmoles)and let react for 4 hours at room temperature. Precipitate theterephthaldehyde monodiethylacetal derivative of glycol chitin (24) with4-6 volumes of ethanol, resuspend the precipitate and wash it with 80%ethanol to remove any excess of reactants. Dissolve the glycol chitinderivative (24) in 40-50 ml of 0.05 M HCl and heat at 100° C. for 20-30minutes to release the aldehyde groups to yield the benzaldehydederivative of glycol chitin (25). The benzaldehyde content of the glycolchitin derivative is determined spectrophotometrically as follows:dissolve 5-8 mg of the glycol chitin derivative in 4 ml of 0.05 M KOHand read the UV spectra between 220 and 320 μm using as a blank asolution of equal concentration of glycol chitin in 0.05 M KOH.Calculate the incorporated benzaldehyde using the extinction coefficientfor this compound determined in 0.05 M KOH. The aldehyde content canalso be determined either spectrophotometrically with MBTH orcalorimetrically with Schiff reagent.

[0103] In another embodiment, described in Scheme 6, glycol chitin (23),formed as described above, is reacted withsuccinimidyl-5-formylsalicylic acid ester (10), which is prepared asfollows. To 0.5 g of 3-formylsalicylic acid (3 mmoles) and 0.42 g of NHS(3.5 mmoles) dissolved in 10-15 ml of DMF, add 1.28 g of CMC (3 mmoles),and let react at room temperature for 5-6 hours protected from moisture.To this reaction mixture add 2.1 ml of p-mercaptoethanol (30 mmoles) toquench the unreacted CMC, mix and react for 10 minutes at roomtemperature and use the succinimidyl-5-formylsalicylic acid esterimmediately. The 5-formylsalicylic acid can be replaced by an equimolaramount of another carboxylated carbonyl-containing compound, such as4,5-dioxoheptanoic acid, 4-formylcinnamic acid, 3-carboxybenzaldehyde or4-formylphenoxyacetic acid). To the glycol chitin (1.7-2 g) dissolved in40 ml of 0.1 M MOBS buffer, pH 7.6, add with stirring the DMF containingthe succinimidyl-5-formylsalicylic acid ester and the mercaptoethanol,and let react with stirring at room temperature for 4-6 hours. The3-formylsalicylic acid derivative of glycol chitin (26) is precipitatedwith 6 volumes of ethanol and washed with 80% ethanol to remove excessreactants. The precipitated glycol chitin derivative is dissolved in50-60 ml of water, dialyzed against water, and lyophilized. The3-formylsalicylic acid content of the glycol chitin derivative isdetermined spectrophotometrically as follows: dissolve 5-8 mg of theglycol chitin derivative in 4 ml of 0.05 M KOH and read the UV spectrabetween 220 and 320 nm using as a blank a solution of equalconcentration of unmodified glycol chitin in 0.05 M KOH. Calculate theincorporated 3-formylsalicylate using the extinction coefficient forthis compound as determined in 0.05 M KOH. The aldehyde content can alsobe determined either colorimetrically with Schiffreagent orspectrophotometrically with MBTH.

[0104] In addition to the procedures described here, other methods, suchas the use of glycidyl ethers, activated halogens, and others, can beused to conjugate carbonyl-containing compounds to the glycosyl residuesof polysaccharides. See, for example, Maron et al., Biochim. Biophys.Acta 278:243 (1972) and Erlanger et al., J. Biol. Chem. 228:713 (1957).

[0105] Other useful carbohydrate-containing compounds that arerecognized by APCs surface receptors include chitins and dextrans whichare of animal and bacterial origin respectively. Examples of suitablecarbohydrate-containing compounds are bacterial teichoic acids and theirderivatives, bacterial lipopolysaccharides, lipid A, and theirderivatives.

[0106] Conjugation of compounds carrying imine-forming carbonyl groupsto non-adjuvant carbohydrate-containing products is contemplated to beuseful in providing intrinsic adjuvanticity to these products and toincrease the efficacy of preventive immunizations. Examples of theseproducts are polysaccharides from streptococci, staphylococci, and otherbacteria that are used as vaccine antigens.

[0107] Pharmaceutical and Veterinary Compositions and Methods of Using

[0108] In a further aspect, a conjugate of the present invention, forexample a compound of Formula I or a physiologically acceptable saltthereof, may be used for the treatment of diseases where there is adefect in the immune system and/or an ineffective host defensemechanism, or to enhance activity to the immune system above normallevels.

[0109] A compound of the invention or a physiologically acceptable saltsthereof may administered for the treatment or prophylaxis ofimmunodeficient mammals alone or combination with other therapeuticagents, for example, with other antiviral agents, or with otheranticancer agents.

[0110] A conjugate of the present invention, for example a compound ofFormula I and physiologically acceptable salts thereof may beadministered for the treatment or prophylaxis of immunodeficient mammalsalone or in combination with other therapeutic agents, for example, withother antiviral agents, or with other anticancer agents.

[0111] Immune adjuvants are compounds which, when administered to anindividual or tested in vitro, increase the immune response to anantigen in a subject or in a test system to which the antigen isadministered.

[0112] By an “effective amount” is meant an amount of a conjugate of thepresent invention that will restore immune function to substantiallynormal levels, or increase immune function above normal levels in orderto eliminate infection.

[0113] By potentiation of an immune response is meant restoration of adepressed immune function, enhancement of a normal immune function, orboth. Immune function is defined as the development and expression ofhumoral (antibody-mediated) immunity, cellular (T-cell-mediated)immunity, or macrophage and granulocyte mediated resistance.

[0114] In this specification the term “immunodeficient patient” isemployed to describe patients with a deficient or defective immunesystem. An immunodeficient patient can be characterized by means of aT-lymphocyte proliferation assay. Using this assay immunodeficientpatients are characterized by a reduced ability of the T-cells torespond to stimulation by mitogens. An example of a mitogen commonlyused in this assay is phytohaemagglutinin (PHA).

[0115] Immunodeficiency and immunosuppression are thought to occur inmany clinical situations where there are lesions in signaling tolymphocytes, particularly T-cells that orchestrate the immune response.T-cells require two signals in order to initiate an effective immuneresponse:

[0116] (i) occupation of the specific T-cell receptor for antigen, and

[0117] (ii) stimulation through costimulatory receptors.

[0118] In the absence of signal (ii), T-cells fail to respond and mayalso become chronically paralyzed or anergic. Persistent viral andbacterial infections and neoplastic disease can produce T-cellhyporesponsiveness by interfering in various ways with the delivery ofsecondary signals and in this way evade the immune response. Theconjugates of the present invention appear to work by substituting orotherwise compensating for deficient costimulatory signals to T-cells.

[0119] Recent studies (Rhodes, J., Immunology Today 17:436 (1996)) haveshown that exogenous Schiff-base-forming compounds can substitute fornatural donors of carbonyl groups and provide a costimulatory signal toCD4 T helper (Th) cells. In a related study (Zheng, B. et al., Science,256:1560 (1992)), treatment of APCs with galactose oxidase to form newaldehyde groups resulted in an adjuvant effect when administered with anantigen to mice.

[0120] These findings stress the role of Schiff-base forming compoundsas stimulators of the immune system. During interaction between an APCand Th-cell there is a transient formation of a Schiff-base between aspecialized APC's carbonyl groups and the Th-cell's amino groups locatedon still undefined cell-surface-receptors. Consequences of theSchiff-base formation are: the biasing of the immune system toward aTh1-type response with an increase in the IL-2 and IFN-γ production inTh-cells, and the enhancement of the CTL response. Schiff-base formingcompounds appear to work by bypassing the co-stimulatory pathwayinvolving the CD-28 receptor on Th-cells and the B7-l receptor presenton APCs.

[0121] There are a variety of circumstances in which the immune systemmay be defective or deficient. For example immune system deficiency iscommon in immature or premature infants (neonates). It may also resultfrom suppression by certain drugs which may be deliberate e.g. as aside-effect of cancer chemotherapy. Disordered growth of one or moreconstituent parts of the immune system, e.g. as in certain forms ofcancer, may also result in immunodeficiency. Immune deficiency can alsobe caused by viral infections, including human immunodeficiency virus(HIV).

[0122] A further aspect of the present invention provides a method oftreating immunodeficient patients, which comprises administering to amammal an effective amount of a conjugate of the present invention, forexample a compound of Formula I, or a physiologically acceptable saltthereof.

[0123] A further aspect of the present invention provides for the use ofa conjugate of the present invention, for example a compound of FormulaI or a physiologically acceptable salt thereof for the treatment and/orprophylaxis of acute and chronic viral infections.

[0124] Examples of acute viruses against which immunopotentiatorytherapy with a conjugate of the present invention, for example acompound of Formula I or a physiologically acceptable salt thereof maybe used, preferably in conjunction with an antiviral agent, are:

[0125] herpes viruses, influenza viruses, parainfluenza viruses,adenoviruses, coxsakie viruses, picorna viruses, rotaviruses, hepatitisA virus, mumps virus, rubella virus, measles virus, pox viruses,respiratory syncytial viruses, papilloma viruses, and enteroviruses,arenavirus, rhinoviruses, poliovirus, Newcastle disease virus, rabiesvirus, and arboviruses.

[0126] Examples of chronic viral infections against whichimmunopotentiatory therapy with conjugates of the present invention maybe used are persistent herpes virus infections, Epstein Barr virusinfection, persistent rubella infections, papillovirus infections,hepatitis virus infections and human immunodeficiency virus infections.

[0127] The conjugates of the invention can be employed alone or incombination with other therapeutic agents for the treatment of the aboveinfections or conditions. Combination therapies according to the presentinvention comprise the administration of at least one conjugate of thepresent invention, for example a compound of the Formula I or aphysiologically acceptable salt thereof and at least one otherpharmaceutically active ingredient. The pharmaceutically activeingredient(s) and compounds of the present invention may be administeredtogether or separately and, when administered separately this may occursimultaneously or sequentially in any order. The amounts of thepharmaceutically active ingredient(s) and compounds of the presentinvention and the relative timings of administration will be selected inorder to achieve the desired combined therapeutic effect. Preferably thecombination therapy involves the administration of one compound of thepresent invention and one of the agents mentioned herein below.

[0128] Examples of such further therapeutic agents include agents thatare effective for the treatment of HIV infections or associatedconditions such as 3′-azido-3′-deoxythymidine (zidovudine), other2′,3′-dideoxynucleosides such as 2′,3′-dideoxycytidine,2′,3′-dideoxyadenosine and 2′,3′-dideoxyinosine, carbovir, acyclicnucleosides (for example, acyclovir), 2′,3′-didehydrothymidine, proteaseinhibitors such asN-tert-butyl-decahydro-2-[-2(R)-hydroxy-4-phenyl-3(S)-[[N-2-quinolylcarbonyl)-L-asparginyl]butyl]-(4aS,8aS)-isoquinoline-3(S)-carboxamide(Ro31-8959), oxathiolan nucleoside analogs such ascis-1-(2-hydroxymethyl)-1,3-oxathiolan-5-yl)-cytosine orcis-1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-5-fluoro-cytosine,3′-deoxy-3′-fluorothymidine, 2′,3′-dideoxy-5-ethynyl-3′-fluorouridine,5-chloro-2′,3′-dideoxy-3′-fluorouridine, Ribavirin,9-[4-hydroxy-2-(hydroxymethyl)but-1-yl]guanine (H2G), TAT inhibitors,such as 7-chloro-5-(2-pyrryl) -3H-1 ,4-benzodiazepin-2(H)-one(Ro5-3335), or 7-chloro-1 ,3-dihydro-5-(1H-pyrrol-2-yl)-3H-1,4-benzodiazepin-2-amine (Ro24-7429) interferons such asα-interferon, renal excretion inhibitors such as probenecid, nucleosidetransport inhibitors such as dipyridamole; pentoxifylline,N-acetylcysteine, precession, α-trichosanthin, phosphonoformic acid, aswell as immunomodulators such as interleukin II, granulocyte macrophagecolony stimulating factors, erythropoetin, soluble CD4 andgenetically-engineered derivatives thereof. Examples of such furthertherapeutic agents which are effective for the treatment of HBVinfections include carbovir, oxathiolan nucleoside analogs such ascis-1-(2-hydroxymethyl)-1,3-oxathiolan-5-yl)cytosine orcis-1-(2-(hydroxymethyl)-1 ,3-oxathiolan-5-yl)-5-fluorocytosine, 2′,3′-didedoxy-5-ethynyl-3′-fluorouridine,5-chloro-2′,3′-didedoxy-3′-fluorouridine, 1-(β-D-arabinofuranosyl)-5-propynyluracil, acyclovir and interferons, such as α-interferon.

[0129] In another aspect the present invention provides the use of aconjugate of the present invention, for example a compound of Formula Ior a physiologically acceptable salt thereof, for the manufacture of amedicament for the treatment and/or prophylaxis of Pneumocystis cariniiinfections in mammals.

[0130] In yet a further aspect the present invention provides for theuse of a conjugate of the present invention, for example a compound ofFormula I or a physiologically acceptable salt thereof to treatconditions resulting from relative T-cell deficiencies such as DiGeorgeSyndrome, fungal infections, mycoplasma infections, tuberculosis,leprosy, and systemic lupus erythemotosus.

[0131] In another aspect of the present invention provides for the useof a conjugate of the present invention, for example a compound ofFormula I or a physiologically acceptable salt thereof for themanufacture of a medicament for the treatment and/or prophylaxis ofcancer in mammals.

[0132] Examples of forms of cancers particularly suitable for treatmentwith compounds the present invention are: melanoma, breast cancer, coloncancer, cancer of the head and neck, gastric cancer, renal cancer,laryngeal cancer, rectal cancer, and non-Hodgkins lymphoma. Cancers thatexpress turnout specific antigens or antigens rarely expressed orexpressed at very low density on normal cells, are likely therapeutictargets. Cancers which contain tumor specific cytotoxic T-cells whichare anergic or otherwise ineffective are likely targets for therapy.Surgically resected tumors where there is a high risk of recurrence arealso suitable for therapy with compounds of the present invention.

[0133] A further aspect of the present invention provides for the use,as a vaccine adjuvant, of a conjugate of the present invention, forexample a compound of Formula I or a physiologically acceptable saltthereof. A vaccine may therefore be prepared by formulating an antigeniccomponent with a conjugate of the present invention.

[0134] Compounds of the present invention may be administered to a humanrecipient by a route selected from oral, parenteral (includingsubcutaneous, intradermal, intramuscular and intravenous), rectal andinhalation. The size of an effective dose of a compound will depend upona number of factors including the identity of the recipient, the type ofimmunopotentiation involved, the severity of the condition to be treatedand the route of administration, and will ultimately be at thediscretion of the attendant physician.

[0135] The effective dose will generally be in the range of 0.03 to 250mg per individual, and most preferably between about 0.05 to about 100mg per dose.

[0136] Immune stimulators are preferably administered only once or twicea week, and in some cases, less frequently. Frequency and length oftreatment vary among species and individuals.

[0137] While it is possible for the compounds of the present inventionto be administered as the raw chemical it is preferable to present themas a pharmaceutical formulation preparation. The formulations of thepresent invention comprise a compound of the present invention, togetherwith one or more acceptable carriers therefor and optionally othertherapeutic ingredients.

[0138] The carrier(s) must be acceptable in the sense of beingcompatible with the other ingredients of the formulation and notdeleterious to the recipient thereof.

[0139] Immune adjuvants are compounds which, when administered to anindividual or tested in vitro, increase the immune response to anantigen in a subject or in a test system to which the antigen isadministered. Some antigens are weakly immunogenic when administeredalone or are toxic to a subject at concentrations that evoke usefulimmune responses in a subject. An immune adjuvant can enhance the immuneresponse of the subject to the antigen by making the antigen morestrongly immunogenic. The adjuvant effect can also result in the abilityto administer a lower dose of antigen to achieve a useful immuneresponse in a subject.

[0140] The immunogen-inducing activity of compounds and compositions ofthe present invention can be determined by a number of known methods.The increase in titer of antibody against a particular antigen uponadministration of a composition of the present invention can be used tomeasure immunogenic activity. (Dalsgaard, K. Acta Veterinia Scandinavica69:1-40 (1978)). One method requires injecting CD-I mice intradermallywith a test composition that includes one or more exogenous antigens.Sera is harvested from mice two weeks later and tested by ELISA foranti-immunogen antibody.

[0141] Compositions of the invention are useful as vaccines to induceactive immunity towards antigens in subjects. Any animal that mayexperience the beneficial effects of the compositions of the presentinvention within the scope of subjects that may be treated. The subjectsare preferably mammals, and more preferably humans.

[0142] Conjugates of the present invention can be employed as a soleadjuvant, or alternatively, can be administered together with otheradjuvants. Such adjuvants useful with the present invention include oiladjuvants (for example, Freund's Complete and Incomplete), saponins,modified saponins, liposomes, mineral salts (for example, AlK(SO₄)₂,AlNa(SO₄)₂, AlNH₄(SO₄), silica, alum, Al(OH)₃, Ca₃(PO₄)₂, kaolin, andcarbon), polynucleotides (for example, poly IC and poly AU acids), andcertain natural substances (for example, wax D from Mycobacteriumtuberculosis, as well as substances found in Corynebacterium parvum,Bordetella pertussis, and members of the genus Brucella), bovine serumalbumin, diphtheria toxoid, tetanus toxoid, edestin, keyhole-limpethemocyanin, Pseudomonal Toxin A, choleragenoid, cholera toxin, pertussistoxin, viral proteins, and eukaryotic proteins such as interferons,interleukins, or tumor necrosis factor. Such proteins may be obtainedfrom natural or recombinant sources according to methods known to thoseskilled in the art. When obtained from recombinant sources, thenon-saponin adjuvant may comprise a protein fragment comprising at leastthe immunogenic portion of the molecule. Other known immunogenicmacromolecules which can be used in the practice of the inventioninclude, but are not limited to, polysaccharides, tRNA,non-metabolizable synthetic polymers such as polyvinylamine,polymethacrylic acid, polyvinylpyrrolidone, mixed polycondensates (withrelatively high molecular weight) of4′,4-diaminodiphenyl-methane-3,3′-dicarboxylic acid and4-nitro-2-aminobenzoic acid (See Sela, M., Science 166:1365-1374 (1969))or glycolipids, lipids or carbohydrates.

[0143] The conjugates of the present invention exhibit adjuvant effectswhen administered over a wide range of dosages and a wide range ofratios to one or more particular antigens being administered.

[0144] The conjugates can be administered either individually or admixedwith other substantially pure adjuvants to achieve an enhancement ofimmune response to an antigen.

[0145] The conjugates of the present invention can be utilized toenhance the immune response to one or more antigens. Typical antigenssuitable for the immune-response provoking compositions of the presentinvention include antigens derived from any of the following: viruses,such as influenza, feline leukemia virus, feline immunodeficiency virus,HIV-1, HIV-2, rabies, measles, hepatitis B, or hoof and mouth disease;bacteria, such as anthrax, diphtheria, Lyme disease, or tuberculosis; orprotozoans, such as Babeosis bovis or Plasmodium. The antigen can beproteins, peptides, polysaccharides, or mixtures thereof. The proteinsand peptides may be purified from a natural source, synthesized by meansof solid phase synthesis, or may be obtained means of recombinantgenetics.

[0146] The conjugates of the present invention can also be administeredalone to potentiate the immune system for treatment of chronicinfectious diseases, especially in immune compromised patients. Examplesof infectious diseases for which conjugates of the present invention canbe employed for therapeutic or prophylactic treatment are described inU.S. Pat. No. 5, 508,310. Potentiation of the immune system by saponinconjugates can also be useful as a preventative measure to limit therisks of nosocomial and/or post-surgery infections.

[0147] Administration of the compounds useful in the method of presentinvention may be by parenteral, intravenous, intramuscular,subcutaneous, intranasal, or any other suitable means. The dosageadministered may be dependent upon the age, weight, kind of concurrenttreatment, if any, and nature of the antigen administered. In general,the saponin/antigen conjugates may be administered over a wide range ofdosages and a wide range of ratios to the antigen being administered.The initial dose may be followed up with a booster dosage after a periodof about four weeks to enhance the immunogenic response. Further boosterdosages may also be administered.

[0148] The conjugates of the present invention may be employed in suchforms as capsules, liquid solutions, emulsions, suspensions or elixirsfor oral administration, or sterile liquid forms such as solutions,emulsions or suspensions. Any inert carrier is preferably used, such assaline, or phosphate-buffered saline, or any such carrier in which thecompounds used in the method of the present invention have suitablesolubility properties for use in the methods of the present invention.

[0149] Having now fully described this invention, it will be understoodto those of ordinary skill in the art that the same can be performedwithin a wide and equivalent range of conditions, formulations, andother parameters without affecting the scope of the invention or anyembodiment thereof. All patents and publications cited herein are fullyincorporated by reference herein in their entirety.

What is claimed is:
 1. A method of enhancing an immune response in ananimal, comprising administering to an animal in need of such enhancinga polysaccharide conjugate in an amount effective to enhance the immuneresponse of said animal, wherein said polysaccharide conjugate consistsessentially of: (i) a polysaccharide which binds to the surface ofAntigen Presenting Cells (APCs) wherein said polysaccharide comprises aminimum of seven saccharides; and (ii) one or more molecules having astable carbonyl group; wherein polysaccharide (i) is attached tomolecules (ii) through (iii) a direct covalent bond or covalently via abifunctional linker in a manner that keeps the stable carbonyl groupintact.
 2. The method of claim 1, wherein said one or more moleculeshaving a stable carbonyl group (ii) are selected from the groupconsisting of aromatic aldehydes, aromatic ketones, cycloalkylaldehydes, cycloalkyl ketones, cycloalkenyl aldehydes, cycloalkenylketones, heterocyclic aldehydes, heterocyclic ketones, heteroaromaticketones, heteroaromatic aldehydes, alkyl aldehyde, alkyl ketones,alkenyl aldehydes and alkenyl ketones.
 3. The method of claim 1, whereinsaid molecules having a stable carbonyl group are bound to saidpolysaccharide via a direct covalent bond.
 4. The method of claim 1,wherein said molecules having a stable carbonyl group are bound to saidpolysaccharide via a residue of a bifunctional linker molecule.
 5. Themethod of claim 1, wherein said polysaccharide is selected from thegroup consisting of β-glucans, mannans, pectic polysaccharides and2-acetamido glucan polysaccharides.
 6. The method of claim 1, whereinsaid molecules having a stable carbonyl group are selected from thegroup consisting of mono- and di-substituted C₆₋₁₀ arylaldehydes, C₆₋₁₀aryl(C₁₋₄)alkylaldehydes, and mixtures thereof.
 7. The method of claim1, wherein said molecules having a stable carbonyl group are phenyl ornaphthyl substituted by a formyl or formyl(C₁₋₄)alkyl substituent, andoptionally including one or two additional substituents independentlyselected from the group consisting of halo, hydroxy, C₁₋₄ alkyl, C₁₋₄alkoxy, trifluoromethyl, and benzyloxy.
 8. The method of claim 1,wherein said molecules having a stable carbonyl group are benzaldehydeand naphthaldehyde, substituted by one or two of hydroxy and halo. 9.The method of claim 1, wherein said molecules having a stable carbonylgroup are 2,3-, 2,4-2,5-, and 3,4-dihydroxybenzaldehyde,5-chloro-2-hydroxybenzaldehyde, vanillin, ethyl vanillin, naringenin, 3-and 4-hydroxybenzaldehyde, or 4-hydroxyphenylacetaldehyde.
 10. Themethod of claim 1, wherein said molecules having a stable carbonyl groupare selected from the group consisting of hydroxy substituted C₁₋₄ alkyl(C₆₋₁₀)aryl ketones, and hydroxy substituted aryl ketones.
 11. Themethod of claim 1, wherein said molecules having a stable carbonyl groupare selected from the group consisting of 2-hydroxyacetophenone,3-hydroxyacetophenone, 4-hydroxyacetophenone, and6-hydroxy-1,2-naphthoquinone.
 12. The method of claim 1, wherein saidmolecules having a stable carbonyl group are selected from the groupconsisting of heteroaryl aldehydes and heteroaryl ketones.
 13. Themethod of claim 1, wherein said molecules having a stable carbonyl groupare thiophene, furan, benzothiophene, benzofuran, pyridine, quinoline,pyridazine, pyrimidine, pyrazole, imidazole, 1,2,3-riazole,1,2,4-triazole, isoxazole, or oxazole, each having a keto, formyl orformyl(C₁₋₄) substituent, and preferably including an additional halo orhydroxy substituent, if these can be accommodated by available ringcarbon atoms.
 14. The method of claim 1, wherein said molecules having astable carbonyl group is one of pyridoxal, 2-thiophenecarboxaldehyde,and 3-thiophenecarboxaldehyde.
 15. The method of claim 4, wherein saidresidue of a bifunctional linker molecule is selected from the groupconsisting of: H₂N—CH₂)_(r)—NH₂, where r is from 2 to 12;HO—CH₂)_(r)—NH₂, where r is from 2 to 12; HS—(CH₂)_(r)—NH₂, where r isfrom 2 to 12; amino acids that are optionally carboxy-protected; andH—(O—CH₂—CH₂)_(n)—H, where n is 1-4.
 16. The method of claim 4, whereinsaid bifunctional linker molecule is selected from the group consistingof: ethylenediamine, 1,4-butanediamine, spermidine, 2,4-diaminobutyricacid, lysine, β-alanine, γ-aminobutyric acid, dialanine, trialanine,3,3′-diaminodipropylamine, diaminopropionic acid,N-(2-aminoethyl)-1,3-propanediamine, and 2-(4-aminophenyl)ethylamine.17. The method of claim 4, wherein said bifunctional linker molecule isselected from the group consisting of: —NH—CH₂)_(r)—NH—, where r is from2-5, —O—CH₂)_(r)—NH—, where r is from 2-5, —NH—CH₂—C(O)—,—O—CH₂—CH₂—O—CH₂—CH₂—, —NH—NH—C(O)—CH₂—, —NH—C(CH₃)₂—C(O)—,—S—(CH₂)_(r)—C(O)—, where r is from 1-5, —S—(CH₂)_(r)—NH—, where r isfrom 2-5, —S—(CH₂)_(r)—O—, where r is from 1-5, —S—CH₂)—CH(NH₂)—C(O)—,—S—(CH₂)—CH(COOH)—NH—, —O—CH₂—CH(OH)—CH₂—S—CH(CO₂H)—NH—,—O—CH₂—CH(OH)—CH₂—S—CH(NH₂)—C(O)—, —O—CH₂—CH(OH)—CH₂—S—CH₂—CH₂—NH—,—S—CH₂—C(O)—NH—CH₂—CH₂—NH—, and —NH—O—C(O)—CH₂—CH₂—O—P(O₂H)—.
 18. Themethod of claim 1, wherein said polysaccharide is selected from thegroup consisting of: β-glucans, mannans, pectic polysaccharides, chitinand its derivatives, murein, bacterial fructans, xanthans, bacterialheteropolysaccharides, fungal pullulan, and esters, sulfonates,sulfates, phosphates; ethers, and cross-linked derivatives thereof. 19.The method of claim 1, wherein said polysaccharide is a β-glucan havinga backbone chain of (1→3)-linked β-D-glucopyranosyl units and which hasβ-D-glucopyranosyl units attached by (1→6) linkages, and a molecularweight of between about 5,000 to about 500,000, and wherein saidβ-glucan is optionally modified by the addition of one or more anionic,cationic or non-ionic groups.
 20. The method of claim 1, wherein saidpolysaccharide is a β-mannans comprising (1→4) polymannose having aterminus reducing mannosyl residue, or the acetylation product thereof.21. The method of claim 1, wherein said polysaccharide is a pecticpolysaccharide selected from the group consisting of homogalacturonans,rhamnogalacturonans, arabans, galactans, and arabinogalactans, andwherein said pectic polysaccharide possesses immunopotentiatingactivity.
 22. A method of potentiating an immune response to an antigenin an animal, comprising administering a polysaccharide conjugate in aneffective amount to potentiate the immune response of said animal tosaid antigen, wherein said polysaccharide conjugate consists essentiallyof: (i) a polysaccharide which binds to the surface of AntigenPresenting Cells (APCs) wherein said polysaccharide comprises a minimumof seven saccharides; and (ii) one or more molecules having a stablecarbonyl group; wherein polysaccharide (i) is attached to molecules (ii)through (iii) a direct covalent bond or covalently via a bifunctionallinker in a manner that keeps the stable carbonyl group intact.
 23. Themethod of claim 22, wherein said one or more molecules having a stablecarbonyl group (ii) are selected from the group consisting of aromaticaldehydes, aromatic ketones, cycloalkyl aldehydes, cycloalkyl ketones,cycloalkenyl aldehydes, cycloalkenyl ketones, heterocyclic aldehydes,heterocyclic ketones, heteroaromatic ketones, heteroaromatic aldehydes,alkyl aldehyde, alkyl ketones, alkenyl aldehydes and alkenyl ketones.24. The method of claim 22, wherein said molecules having a stablecarbonyl group are bound to said polysaccharide via a direct covalentbond.
 25. The method of claim 22, wherein said molecules having a stablecarbonyl group are bound to said polysaccharide via a residue of abifunctional linker molecule.
 26. The method of claim 22, wherein saidpolysaccharide is selected from the group consisting of β-glucans,mannans, pectic polysaccharides and 2-acetamido glucan polysaccharides.27. The method of claim 22, wherein said molecules having a stablecarbonyl group are selected from the group consisting of mono- anddi-substituted C₆₋₁₀ arylaldehydes, C₆₋₁₀ aryl(C₁₋₄)alkylaldehydes, andmixtures thereof.
 28. The method of claim 22, wherein said moleculeshaving a stable carbonyl group are phenyl or naphthyl substituted by aformyl or formyl(C₁₋₄)alkyl substituent, and optionally including one ortwo additional substituents independently selected from the groupconsisting of halo, hydroxy, C₁₋₄ alkyl, C₁₋₄ alkoxy, trifluoromethyl,and benzyloxy.
 29. The method of claim 22, wherein said molecules havinga stable carbonyl group are benzaldehyde and naphthaldehyde, substitutedby one or two of hydroxy and halo.
 30. The method of claim 22, whereinsaid molecules having a stable carbonyl group are 2,3-, 2,4-2,5-, and3,4-dihydroxybenzaldehyde, 5-chloro-2-hydroxybenzaldehyde, vanillin,ethyl vanillin, naringenin, 3- and 4-hydroxybenzaldehyde, or4-hydroxyphenylacetaldehyde.
 31. The method of claim 22, wherein saidmolecules having a stable carbonyl group are selected from the groupconsisting of hydroxy substituted C₁₋₄alkyl (C₆₋₁₀)aryl ketones, andhydroxy substituted aryl ketones.
 32. The method of claim 22, whereinsaid molecules having a stable carbonyl group are selected from thegroup consisting of 2-hydroxyacetophenone, 3-hydroxyacetophenone,4-hydroxyacetophenone, and 6-hydroxy-1,2-naphthoquinone.
 33. The methodof claim 22, wherein said molecules having a stable carbonyl group areselected from the group consisting of heteroaryl aldehydes andheteroaryl ketones.
 34. The method of claim 22, wherein said moleculeshaving a stable carbonyl group are thiophene, furan, benzothiophene,benzofuran, pyridine, quinoline, pyridazine, pyrimidine, pyrazole,imidazole, 1,2,3-triazole, 1,2,4-triazole, isoxazole, or oxazole, eachhaving a keto, formyl or formyl(C₁₋₄) substituent, and preferablyincluding an additional halo or hydroxy substituent, if these can beaccommodated by available ring carbon atoms.
 35. The method of claim 22,wherein said molecules having a stable carbonyl group is one ofpyridoxal, 2-thiophenecarboxaldehyde, and 3-thiophenecarboxaldehyde. 36.The method of claim 25, wherein said residue of a bifunctional linkermolecule is selected from the group consisting of: H₂N—(CH₂)_(r)—NH₂,where r is from 2 to 12; HO—(CH₂)_(r)—NH₂, where r is from 2 to 12;HS—(CH₂)_(r)—NH₂, where r is from 2 to 12; amino acids that areoptionally carboxy-protected; and H—(O—CH₂—CH₂)_(n)—OH, where n is 1-4.37. The method of claim 25, wherein said bifunctional linker molecule isselected from the group consisting of: ethylenediamine,1,4-butanediamine, spermidine, 2,4-diaminobutyric acid, lysine,β-alanine, γ-aminobutyric acid, dialanine, trialanine,3,3′-diaminodipropylamine, diaminopropionic acid,N-(2-aminoethyl)-1,3-propanediamine, and 2-(4-aminophenyl)ethylamine.38. The method of claim 25, wherein said bifunctional linker molecule isselected from the group consisting of: —NH—(CH₂)_(r)—NH—, where r isfrom 2-5, —O—(CH₂)_(r)=NH—, where r is from 2-5, —NH—CH₂—C(O)—,—O—CH₂—CH₂—O—CH₂—CH₂—O—, —NH—NH—C(O)—CH₂—, —NH—C(CH₃)₂—C(O)—,—S—(CH₂)_(r)—C(O)—, where r is from 1-5, —S—(CH₂)_(r)—NH—, where r isfrom 2-5, —S—(CH₂)_(r)—O—, where r is from 1-5, —S—(CH₂)—CH(NH₂)—C(O)—,—S—(CH₂)—CH(COOH)—NH—, —O—CH₂—CH(OH)—CH₂—S—CH(CO₂H)—NH—,—OCH₂—CH(OH)—CH₂—S—CH(NH₂)—C(O)—, —O—CH₂—CH(OH)—CH₂—S—CH₂—CH₂—NH—,—S—CH₂—C(O)—NH—CH₂—CH₂—NH—, and —NH—O—C(O)—CH₂—CH₂—O—P(O₂H)—.
 39. Themethod of claim 22, wherein said polysaccharide is selected from thegroup consisting of: β-glucans, mannans, pectic polysaccharides, chitinand its derivatives, murein, bacterial fructans, xanthans, bacterialheteropolysaccharides, fungal pullulan, and esters, sulfonates,sulfates, phosphates; ethers, and cross-linked derivatives thereof. 40.The method of claim 22, wherein said polysaccharide is a β-glucan havinga backbone chain of (1→3)-linked β-D-glucopyranosyl units and which hasβ-D-glucopyranosyl units attached by (1→6) linkages, and a molecularweight of between about 5,000 to about 500,000, and wherein saidβ-glucan is optionally modified by the addition of one or more anionic,cationic or non-ionic groups.
 41. The method of claim 22, wherein saidpolysaccharide is a β-mannans comprising (1→4) polymannose having aterminus reducing mannosyl residue, or the acetylation product thereof.42. The method of claim 22, wherein said polysaccharide is a pecticpolysaccharide selected from the group consisting of homogalacturonans,rhamnogalacturonans, arabans, galactans, and arabinogalactans, andwherein said pectic polysaccharide possesses immunopotentiatingactivity.
 43. A method of vaccinating an animal, comprisingadministering a polysaccharide conjugate to said animal, wherein saidpolysaccharide conjugate consists essentially of: (i) a polysaccharidecapable of binding to the cell surface of Antigen Presenting Cells(APCs) wherein said polysaccharide comprises a minimum of sevensaccharides; and (ii) one or more molecules having a stable carbonylgroup; wherein polysaccharide (i) is attached to molecules (ii) through(iii) a direct covalent bond or covalently via a bifunctional linker ina manner that keeps the stable carbonyl group intact.
 44. A method oftreating a disease in an animal comprising administering an effectiveamount of a polysaccharide conjugate, wherein said polysaccharideconjugate consists essentially of: (i) a polysaccharide which binds tothe surface of Antigen Presenting Cells (APCs) wherein saidpolysaccharide comprises a minimum of seven saccharides; and (ii) one ormore molecules having a stable carbonyl group; wherein polysaccharide(i) is attached to molecules (ii) through (iii) a direct covalent bondor covalently via a bifunctional linker in a manner that keeps thestable carbonyl group intact.
 45. The method of claim 44 furthercomprising the co-administration of at least one therapeutic agent oradjuvant.
 46. The method of claim 44 or 45 wherein said disease is acancer or a bacterial or viral infection or is the result of cancer or abacterial or viral infection.
 47. The method of claim 46 wherein saidviral infection is the result of a virus selected from the groupconsisting of: herpes, Epstein Barr, rubella, papillovirus, and humanimmunodeficiency virus.
 48. The method of claim 46 wherein said diseaseis hepatitis.
 49. The method of claim 46 wherein said cancer is selectedfrom the group consisting of: non-Hodgkins lymphoma, melanoma, breast,colon, head and neck, gastric, renal, laryngeal and rectal cancers,cancers that express antigens, cancers that contain tumor specificcytotoxic T-cells which are anergic, and cancers that have beensurgically resected where there is a risk of recurrence.
 50. The methodof claim 46 wherein said disease is a Pneumocystis carinii infection.51. The method of claim 44 or 45 wherein said disease is selected fromthe group consisting of: DiGeorge Syndrome, fungal infections,mycoplasma infections, tuberculosis, leprosy and systemic lupuserythemotosus.
 52. The method of claim 45 wherein said adjuvant is anantigen.
 53. The method of claim 51 wherein said antigen is derived froma virus, bacteria or protozoan.
 54. The method of claim 52 wherein saidantigen is derived from one of the following: influenza, felineleukemia, feline immunodeficiency virus, HIV-1, HIV-2, rabies, measles,hepatitis B, hoof and mouth disease, anthrax, diptheria, Lyme disease,tuberculosis, Babeosis bovis or Plasmodium.
 55. The method of claim 45wherein said therapeutic agent is an antiviral or anticancer agent. 56.The method of claim 55 wherein said antiviral agent is an agent for thetreatment of viruses selected from the group consisting of: herpes,influenza, parainfluenza, adenoviruses, coxsakie, picorna, rotaviruses,hepatitis A, mumps, rubella, measles, pox, respiratory synctial,papilloma, enteroviruses, arenaviruses, rhinoviruses, polio, Newcastledisease, rabies and arboviruses.
 57. The method of claim 55 wherein saidantiviral agent is selected from the the group consisting of:3′-azido-3′-deoxythymidine, 2′,3′-dideoxycytidine,2′,3′-dideoxyadenosine, 2′,3′-dideoxyinosine, carbovir,2′,3′-didehydrothymidine,N-tert-butyl-decahydro-2-[-2(R)-hydroxy-4-phenyl-3(S)-[[N-2-quinolylcarbonyl)-L-asparginyl]butyl]-(4aS,8aS)-isoquinoline-3(S)-carboxamide,cis-1-(2-hydroxymethyl)-1,3-oxathiolan-5-yl)-cytosine orcis-1-(2-(hydroxymethyl) -1 ,3-oxathiolan-5-yl)-5-fluoro-cytosine,3′-deoxy-3′-fluorothymidine, 2′,3′-dideoxy-5-ethynyl-3′-fluorouridine,5-chloro-2′,3′-dideoxy-3′-fluorouridine, Ribavirin,9-[4-hydroxy-2-(hydroxymethyl)but-1-yl]guanine,7-chloro-5-(2-pyrryl)-3H-1,4-benzodiazepine-2(H)-one,7-chloro-1,3-dihydro-5-(1H-pyrrol-2-yl) -3H-1,4-benzodiazepin-2-amine,α-interferon, probenecid, dipyridamole; pentoxifylline,N-acetylcysteine, precession, α-trichosanthin, phosphonoformic acid,interleukin II, erythropoetin, and1-(β-D-arabinofuranosyl)-5-propynyluracil.
 58. The method of claim 45wherein said adjuvant is selected from the group consisting of: oiladjuvants, saponins, modified saponins, liposomes, mineral salts,polynucleotides, certain natural substances isolated from Mycobacteriumtuberculosis, Corynebacterium parvum, Bordetella pertussis or members ofthe genus Brucella, bovine serum, albumin, diptheria toroid, tetanustoroid, edestin, keyhole-limpet hemocyanin, Pseudomonal Toxin A,choleragenoid, cholera toxin, pertussis toxin, viral proteins,eukaryotic proteins, polysaccharide, RNA, polyvinylamine,polymethacrylic acid, polyvinylpyrrolidone, polycondensates,glycolipids, lipids and carbohydrates.